library(here)
library(tidyverse)
library(cowplot)
library(GGally)
theme_set(theme_light())

library(scuttle)
library(scater)

library(kableExtra)
library(SCandwichCompanion)

Setup

## Directory setup
here_root <- "benchmarks/lupus"
here::i_am(file.path(here_root, "analysis/lupus-mock-results.Rmd"))
#> here() starts at /Users/milan/Projects/SCandwich-analyses

res_dir <- here::here(here_root, "results")
fig_dir <- here::here(here_root, "figures")
  • Using methods: muscat, sandwichTtestLRPrior1, pseudobulkSandwichTtest, NBpseudobulkSandwichTtest
  • Using celltypes: combined

Load results

res_files <- map(celltypes, ~ get_mock_res_files(
    dataset = "lupus",
    methods = methods, celltype = .x
)) %>%
    set_names(celltypes)

res_list <- map_depth(res_files, 2, readRDS)

## Do some wrangling when using 'combined' celltypes
if (combined) {
    ## Only 1 top-level entry + transpose so that celltypes are the top-level
    res_list <- transpose(res_list[[1]])
}

## Check for expected data structure
stopifnot(unlist(map_depth(res_list, 3, ~ is.data.frame(.x$results))))

Load SCE objects

data_files <- map(celltypes, ~ get_SCE_files(
    dataset = "lupus", which = "mock_replicates",
    celltype = .x
)) %>%
    set_names(celltypes)
sce_objects <- map(data_files, readRDS)

Data overview

  • The results were generated from 5 mock replicates
  • Each replicate was generated by randomly splitting the subjects in two mock groups
  • No sub-sampling of cells per patient was performed for this data
map_dfr(sce_objects,
    ~ map_dfr(.x, function(x) c(nrows = nrow(x), ncols = ncol(x)),
        .id = "replicate"
    ),
    .id = "celltypes"
)

Subjects are divided across mock groups as follows:

map(sce_objects[[1]], function(x) table(x$ind_cov, x$mock_group))
#> $replicate_1
#>                    
#>                        A    B
#>   IGTB141_IGTB141    964    0
#>   IGTB143_IGTB143   1417    0
#>   IGTB195_IGTB195      0 1571
#>   IGTB469_IGTB469   1263    0
#>   IGTB498_IGTB498   1594    0
#>   IGTB508_IGTB508      0 1317
#>   IGTB514_IGTB514   1333    0
#>   IGTB645_IGTB645      0 1299
#>   IGTB670_IGTB670   1290    0
#>   IGTB826_IGTB826      0 1154
#>   IGTB884_IGTB884      0  967
#>   IGTB986_IGTB986    926    0
#>   IGTB1372_IGTB1372    0  836
#>   IGTB1506_IGTB1506    0  927
#>   IGTB1539_IGTB1539    0  818
#>   IGTB1575_IGTB1575  823    0
#>   IGTB1650_IGTB1650 1238    0
#>   IGTB1762_IGTB1762 1815    0
#>   IGTB1768_IGTB1768 1545    0
#>   IGTB1789_IGTB1789    0 1105
#>   IGTB1793_IGTB1793 1233    0
#>   IGTB1814_IGTB1814    0 1400
#>   IGTB1815_IGTB1815 1063    0
#>   IGTB1826_IGTB1826    0  974
#>   IGTB1827_IGTB1827 1117    0
#>   IGTB1828_IGTB1828  959    0
#>   IGTB1840_IGTB1840 1422    0
#>   IGTB1844_IGTB1844 1252    0
#>   IGTB1871_IGTB1871    0  973
#>   IGTB1895_IGTB1895    0 1098
#>   IGTB1901_IGTB1901    0  960
#>   IGTB1906_IGTB1906  779    0
#>   IGTB1908_IGTB1908    0  773
#>   IGTB1913_IGTB1913    0 1659
#>   IGTB1916_IGTB1916    0  985
#>   IGTB1921_IGTB1921 1468    0
#>   IGTB1952_IGTB1952    0 1263
#>   IGTB1966_IGTB1966    0 1782
#>   IGTB1974_IGTB1974  957    0
#>   IGTB1982_IGTB1982    0  827
#>   IGTB1987_IGTB1987    0 1526
#>   IGTB1996_IGTB1996    0  622
#>   IGTB2007_IGTB2007 1001    0
#>   IGTB2065_IGTB2065  954    0
#> 
#> $replicate_2
#>                    
#>                        A    B
#>   IGTB141_IGTB141      0  964
#>   IGTB143_IGTB143   1417    0
#>   IGTB195_IGTB195   1571    0
#>   IGTB469_IGTB469      0 1263
#>   IGTB498_IGTB498   1594    0
#>   IGTB508_IGTB508   1317    0
#>   IGTB514_IGTB514      0 1333
#>   IGTB645_IGTB645      0 1299
#>   IGTB670_IGTB670      0 1290
#>   IGTB826_IGTB826   1154    0
#>   IGTB884_IGTB884    967    0
#>   IGTB986_IGTB986      0  926
#>   IGTB1372_IGTB1372    0  836
#>   IGTB1506_IGTB1506    0  927
#>   IGTB1539_IGTB1539  818    0
#>   IGTB1575_IGTB1575    0  823
#>   IGTB1650_IGTB1650    0 1238
#>   IGTB1762_IGTB1762 1815    0
#>   IGTB1768_IGTB1768 1545    0
#>   IGTB1789_IGTB1789    0 1105
#>   IGTB1793_IGTB1793    0 1233
#>   IGTB1814_IGTB1814    0 1400
#>   IGTB1815_IGTB1815 1063    0
#>   IGTB1826_IGTB1826  974    0
#>   IGTB1827_IGTB1827 1117    0
#>   IGTB1828_IGTB1828    0  959
#>   IGTB1840_IGTB1840 1422    0
#>   IGTB1844_IGTB1844 1252    0
#>   IGTB1871_IGTB1871    0  973
#>   IGTB1895_IGTB1895 1098    0
#>   IGTB1901_IGTB1901  960    0
#>   IGTB1906_IGTB1906  779    0
#>   IGTB1908_IGTB1908  773    0
#>   IGTB1913_IGTB1913    0 1659
#>   IGTB1916_IGTB1916    0  985
#>   IGTB1921_IGTB1921 1468    0
#>   IGTB1952_IGTB1952    0 1263
#>   IGTB1966_IGTB1966 1782    0
#>   IGTB1974_IGTB1974    0  957
#>   IGTB1982_IGTB1982  827    0
#>   IGTB1987_IGTB1987    0 1526
#>   IGTB1996_IGTB1996    0  622
#>   IGTB2007_IGTB2007    0 1001
#>   IGTB2065_IGTB2065  954    0
#> 
#> $replicate_3
#>                    
#>                        A    B
#>   IGTB141_IGTB141      0  964
#>   IGTB143_IGTB143      0 1417
#>   IGTB195_IGTB195   1571    0
#>   IGTB469_IGTB469      0 1263
#>   IGTB498_IGTB498   1594    0
#>   IGTB508_IGTB508   1317    0
#>   IGTB514_IGTB514      0 1333
#>   IGTB645_IGTB645   1299    0
#>   IGTB670_IGTB670      0 1290
#>   IGTB826_IGTB826   1154    0
#>   IGTB884_IGTB884    967    0
#>   IGTB986_IGTB986    926    0
#>   IGTB1372_IGTB1372  836    0
#>   IGTB1506_IGTB1506  927    0
#>   IGTB1539_IGTB1539    0  818
#>   IGTB1575_IGTB1575    0  823
#>   IGTB1650_IGTB1650    0 1238
#>   IGTB1762_IGTB1762    0 1815
#>   IGTB1768_IGTB1768 1545    0
#>   IGTB1789_IGTB1789 1105    0
#>   IGTB1793_IGTB1793 1233    0
#>   IGTB1814_IGTB1814    0 1400
#>   IGTB1815_IGTB1815 1063    0
#>   IGTB1826_IGTB1826    0  974
#>   IGTB1827_IGTB1827    0 1117
#>   IGTB1828_IGTB1828    0  959
#>   IGTB1840_IGTB1840 1422    0
#>   IGTB1844_IGTB1844    0 1252
#>   IGTB1871_IGTB1871  973    0
#>   IGTB1895_IGTB1895    0 1098
#>   IGTB1901_IGTB1901    0  960
#>   IGTB1906_IGTB1906  779    0
#>   IGTB1908_IGTB1908    0  773
#>   IGTB1913_IGTB1913 1659    0
#>   IGTB1916_IGTB1916  985    0
#>   IGTB1921_IGTB1921 1468    0
#>   IGTB1952_IGTB1952 1263    0
#>   IGTB1966_IGTB1966    0 1782
#>   IGTB1974_IGTB1974  957    0
#>   IGTB1982_IGTB1982    0  827
#>   IGTB1987_IGTB1987    0 1526
#>   IGTB1996_IGTB1996  622    0
#>   IGTB2007_IGTB2007    0 1001
#>   IGTB2065_IGTB2065  954    0
#> 
#> $replicate_4
#>                    
#>                        A    B
#>   IGTB141_IGTB141      0  964
#>   IGTB143_IGTB143      0 1417
#>   IGTB195_IGTB195      0 1571
#>   IGTB469_IGTB469   1263    0
#>   IGTB498_IGTB498      0 1594
#>   IGTB508_IGTB508   1317    0
#>   IGTB514_IGTB514   1333    0
#>   IGTB645_IGTB645      0 1299
#>   IGTB670_IGTB670      0 1290
#>   IGTB826_IGTB826   1154    0
#>   IGTB884_IGTB884    967    0
#>   IGTB986_IGTB986    926    0
#>   IGTB1372_IGTB1372  836    0
#>   IGTB1506_IGTB1506    0  927
#>   IGTB1539_IGTB1539  818    0
#>   IGTB1575_IGTB1575    0  823
#>   IGTB1650_IGTB1650 1238    0
#>   IGTB1762_IGTB1762    0 1815
#>   IGTB1768_IGTB1768    0 1545
#>   IGTB1789_IGTB1789    0 1105
#>   IGTB1793_IGTB1793    0 1233
#>   IGTB1814_IGTB1814 1400    0
#>   IGTB1815_IGTB1815 1063    0
#>   IGTB1826_IGTB1826  974    0
#>   IGTB1827_IGTB1827    0 1117
#>   IGTB1828_IGTB1828    0  959
#>   IGTB1840_IGTB1840 1422    0
#>   IGTB1844_IGTB1844 1252    0
#>   IGTB1871_IGTB1871  973    0
#>   IGTB1895_IGTB1895 1098    0
#>   IGTB1901_IGTB1901    0  960
#>   IGTB1906_IGTB1906  779    0
#>   IGTB1908_IGTB1908    0  773
#>   IGTB1913_IGTB1913    0 1659
#>   IGTB1916_IGTB1916    0  985
#>   IGTB1921_IGTB1921    0 1468
#>   IGTB1952_IGTB1952    0 1263
#>   IGTB1966_IGTB1966 1782    0
#>   IGTB1974_IGTB1974  957    0
#>   IGTB1982_IGTB1982    0  827
#>   IGTB1987_IGTB1987    0 1526
#>   IGTB1996_IGTB1996  622    0
#>   IGTB2007_IGTB2007 1001    0
#>   IGTB2065_IGTB2065    0  954
#> 
#> $replicate_5
#>                    
#>                        A    B
#>   IGTB141_IGTB141      0  964
#>   IGTB143_IGTB143      0 1417
#>   IGTB195_IGTB195   1571    0
#>   IGTB469_IGTB469      0 1263
#>   IGTB498_IGTB498   1594    0
#>   IGTB508_IGTB508      0 1317
#>   IGTB514_IGTB514   1333    0
#>   IGTB645_IGTB645   1299    0
#>   IGTB670_IGTB670      0 1290
#>   IGTB826_IGTB826   1154    0
#>   IGTB884_IGTB884    967    0
#>   IGTB986_IGTB986      0  926
#>   IGTB1372_IGTB1372  836    0
#>   IGTB1506_IGTB1506    0  927
#>   IGTB1539_IGTB1539    0  818
#>   IGTB1575_IGTB1575  823    0
#>   IGTB1650_IGTB1650    0 1238
#>   IGTB1762_IGTB1762    0 1815
#>   IGTB1768_IGTB1768    0 1545
#>   IGTB1789_IGTB1789    0 1105
#>   IGTB1793_IGTB1793 1233    0
#>   IGTB1814_IGTB1814 1400    0
#>   IGTB1815_IGTB1815 1063    0
#>   IGTB1826_IGTB1826  974    0
#>   IGTB1827_IGTB1827 1117    0
#>   IGTB1828_IGTB1828  959    0
#>   IGTB1840_IGTB1840 1422    0
#>   IGTB1844_IGTB1844    0 1252
#>   IGTB1871_IGTB1871  973    0
#>   IGTB1895_IGTB1895 1098    0
#>   IGTB1901_IGTB1901    0  960
#>   IGTB1906_IGTB1906  779    0
#>   IGTB1908_IGTB1908  773    0
#>   IGTB1913_IGTB1913 1659    0
#>   IGTB1916_IGTB1916    0  985
#>   IGTB1921_IGTB1921    0 1468
#>   IGTB1952_IGTB1952    0 1263
#>   IGTB1966_IGTB1966    0 1782
#>   IGTB1974_IGTB1974    0  957
#>   IGTB1982_IGTB1982    0  827
#>   IGTB1987_IGTB1987 1526    0
#>   IGTB1996_IGTB1996  622    0
#>   IGTB2007_IGTB2007    0 1001
#>   IGTB2065_IGTB2065    0  954

Extract results of interest

Runtimes

## Get runtimes for each celltype
runtimes <- map(res_list, ~ map_dfr(.x, get_runtimes, depth = 1, .id = "method"))

P-values

res_tables <- map_depth(
    res_list, 2,
    ~ get_aggregated_rep_tables(.x, depth = 1)
) %>%
    map(combine_tables, .id = "method")

Visualize results

Run times

runtime_plots <- imap(runtimes,
    ~ plot_run_times(.x, width = 0.2, height = 0) +
      ggtitle(.y)
)

B_mem

ncM

T4_naive

Number of significant genes for different FDR levels

n_sign_summaries <- map(res_tables, function(tmp) {
    tmp %>%
        group_by(replicate, method) %>%
        summarise(
            sum(FDR < 0.01),
            sum(FDR < 0.05),
            sum(FDR < 0.1),
            .groups = "drop"
        )
})

B_mem

Cells per patient: B_mem
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 2
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 3
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 1
sandwichTtestLRPrior1 0 0 1
Replicate: 4
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 5
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0

ncM

Cells per patient: ncM
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 2
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 3
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 1
Replicate: 4
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 5
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0

T4_naive

Cells per patient: T4_naive
method sum(FDR < 0.01) sum(FDR < 0.05) sum(FDR < 0.1)
Replicate: 1
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 2
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 3
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 4
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0
Replicate: 5
muscat NA NA NA
NBpseudobulkSandwichTtest 0 0 0
pseudobulkSandwichTtest 0 0 0
sandwichTtestLRPrior1 0 0 0

P-value distributions

pval_figs <- map(res_tables, pval_hist)

B_mem

#> Warning: Removed 482 rows containing non-finite values (stat_bin).

ncM

#> Warning: Removed 482 rows containing non-finite values (stat_bin).

T4_naive

#> Warning: Removed 482 rows containing non-finite values (stat_bin).

P-value distributions stratified by expression level

For each replicate, calculate average log-normalized counts, using scuttle::calculateAverage().

## Calculate average counts for each replicate
avg_counts <- map_depth(sce_objects, 2,
    ~ calculateAverage(counts(.x))
)

## Split res_tables per replicate
res_tables_split <- map(res_tables, ~ split(.x, .x$replicate))

## Add average counts to results tables
## Note that for the 'combined' celltypes, the average counts will be the same
## for each celltype contrast
res_tables_split <- map2(
    res_tables_split, avg_counts,
    ~ map2(.x, .y, function(res, avg_cnt) {
        res$avg_count <- avg_cnt[res$gene]
        res
    })
)

Make p-value histograms stratified by the average counts.

pval_hist_strat_plots <- map(res_tables_split,
    ~ imap(.x, function(y, replicate) {
        replicate <- str_remove(replicate, "replicate_")
        title <- paste("Replicate:", replicate)
        pval_hist_strat(y, n_groups = 6) +
            ggtitle(title)
    })
)

B_mem

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

ncM

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

T4_naive

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 88 rows containing non-finite values (stat_bin).

#> Warning: Removed 102 rows containing non-finite values (stat_bin).

Session info

Session info
#> [1] "2022-05-30 17:04:12 CEST"
#> Local:    lupus-data /Users/milan/Projects/SCandwich-analyses
#> Remote:   lupus-data @ origin (git@github.com:statOmics/SCandwich-analyses.git)
#> Head:     [2f83457] 2022-05-19: Enable combined celltype analyses again
#> ─ Session info  🇲🇱  🤛🏿  🤷🏼   ───────────────────────────────────────────────────────────────────────────────────────
#>  hash: flag: Mali, left-facing fist: dark skin tone, person shrugging: medium-light skin tone
#> 
#>  setting  value
#>  version  R version 4.1.3 (2022-03-10)
#>  os       macOS Big Sur/Monterey 10.16
#>  system   x86_64, darwin17.0
#>  ui       X11
#>  language en_US.UTF-8
#>  collate  en_US.UTF-8
#>  ctype    en_US.UTF-8
#>  tz       Europe/Brussels
#>  date     2022-05-30
#>  pandoc   2.17.1.1 @ /usr/local/bin/ (via rmarkdown)
#> 
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